Immunogenic fusion proteins against infectious animal diseases

Abstract

Immunogenic fusion proteins against infectious animal diseases. A fusion protein is disclosed, which comprises a CD40-binding domain; an antigen of a pathogen; a translocation domain located between the CD40-binding domain and the antigen, and a furin and/or cathepsin L cleavage site located between the CD40-binding domain and the translocation domain. Also disclosed are pharmaceutical compositions, expression vectors and use of the fusion proteins of the invention for eliciting an antigen-specific cell-mediated immune response, or for reducing, inhibiting, treating and/or ameliorating an infectious animal disease caused by a pathogen in an animal in need thereof.

1 . A fusion protein comprising:
 (a) a CD40-binding domain, which is a CD40 ligand (CD40L) or a functional fragment thereof comprising an amino acid sequence that is at least 95% identical to SEO ID NO: 19, said CD40L or functional fragment thereof consisting of 154-261 amino acid residues in length;   (b) an antigen of a pathogen;   (c) a translocation domain, located between the CD40-binding domain and the antigen, said translocation domain being selected from the group consisting of:
 (c1) a Shiga toxin (Stx) translocation peptide; and 
 (c2) a  Pseudomonas  Exotoxin A (PE) translocation peptide; and 
   (d) a furin and/or cathepsin L cleavage site, located between the CD40-binding domain and the translocation domain,   wherein the pathogen is at least one selected from the group consisting of Classical Swine Fever Virus (CSFV), African Swine Fever Virus (ASFV), Porcine Circovirus 2 (PCV2), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Epidemic Diarrhea Virus (PEDV), Foot-and-Mouth Disease Virus (FMDV), Swine Vesicular Disease Virus (SVDV), Pseudorabies Virus (PRV), Transmissible Gastroenteritis Virus (TGEV),  Mycoplasma hyopneumoniae , Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), Infectious Bursal Disease Virus (IBDV), Parvovirus, Poxvirus, Rotavirus, and Influenza Virus; and further wherein when the translocation domain is the Stx translocation peptide, the antigen is located at the N-terminal of the fusion protein; and when the translocation domain is the PE translocation peptide, the CD40-binding domain is located at the N-terminal of the fusion protein.   
     
     
         2 . The fusion protein of  claim 1 , wherein the translocation domain is the PE translocation peptide, the PE translocation peptide consisting of 26-112 amino acid residues in length and comprising an amino acid sequence of SEO ID NO: 5. 
     
     
         3 . The fusion protein of  claim 1 , wherein the translocation domain is the PE translocation peptide consisting of 26-112 amino acid residues in length and comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 5, 6, 7, 8 or 9. 
     
     
         4 . The fusion protein of  claim 1 , wherein the translocation domain is the Stx translocation peptide, the Stx translocation peptide consisting of 8-84 amino acid residues in length and comprising an amino acid sequence of SEQ ID NOs:12. 
     
     
         5 . The fusion protein of  claim 1 , wherein the translocation domain is the Stx translocation peptide consisting of 8-84 amino acid residues in length and comprises an amino acid sequence that is at least 95% identical to SEQ ID NOs: 12, 13, 14, 15 or 16. 
     
     
         6 . The fusion protein of  claim 1 , wherein the furin and/or cathepsin L cleavage site comprises an amino acid sequence of SEQ ID NO: 1 or 2. 
     
     
         7 . The fusion protein of  claim 1 , further comprising a peptide linker, said peptide linker comprising the furin and/or cathepsin L cleavage site located between the CD40-binding domain and the translocation domain. 
     
     
         8 . The fusion protein of  claim 1 , wherein the CD40L or the functional fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 17, 18 and 19. 
     
     
         9 . The fusion protein of  claim 1 , wherein the CD40L consists of 154-261 amino acid residues in length and comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 17, 18 or 19. 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . The fusion protein of  claim 2 , further comprising an endoplasmic reticulum (ER) retention sequence located at the C-terminus of the antigen. 
     
     
         13 . The fusion protein of  claim 3 , further comprising a CD28-activating peptide located between the CD40-binding domain and the furin and/or cathepsin L cleavage site, wherein the CD28-activating peptide has a length of 28-53 amino acid residues and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 35, 36 and 37. 
     
     
         14 . A pharmaceutical composition comprising:
 (a) the fusion protein of  claim 1 ; and   (b) a pharmaceutical acceptable carrier and/or an adjuvant.   
     
     
         15 . A method for eliciting an antigen-specific, cell-mediated immune response, or for reducing, inhibiting, treating, and/or ameliorating an infectious animal disease caused by a pathogen in an animal in need thereof, comprising:
 administering a therapeutically effective amount of the fusion protein of  claim 1  to the animal in need thereof, and thereby eliciting the antigen-specific, cell-mediated immune response, or reducing, inhibiting, treating, and/or ameliorating the infectious animal disease caused by the pathogen in the animal in need thereof.   
     
     
         16 . The fusion protein of  claim 3 , wherein the PE translocation peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8 and 9. 
     
     
         17 . The fusion protein of  claim 5 , wherein the Stx translocation peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15 and 16. 
     
     
         18 . A fusion protein comprising:
 (a) a CD40-binding domain, which is a CD40 ligand (CD40L) or a functional fragment thereof comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 19, said CD40L or functional fragment thereof consisting of 154-261 amino acid residues in length;   (b) an antigen of a pathogen;   (c) a translocation domain, located between the CD40-binding domain and the antigen, said translocation domain being selected from the group consisting of:
 (c1) a Shiga toxin (Stx) translocation peptide, comprising an amino acid sequence of SEQ ID NO: 12; and 
 (c2) a  Pseudomonas  Exotoxin A (PE) translocation peptide, comprising an amino acid sequence of SEQ ID NO: 5; and 
   (d) a furin and/or cathepsin L cleavage site, located between the CD40-binding domain and the translocation domain,   wherein the pathogen is at least one selected from the group consisting of Classical Swine Fever Virus (CSFV), African Swine Fever Virus (ASFV), Porcine Circovirus 2 (PCV2), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Epidemic Diarrhea Virus (PEDV), Foot-and-Mouth Disease Virus (FMDV), Swine Vesicular Disease Virus (SVDV), Pseudorabies Virus (PRV), Transmissible Gastroenteritis Virus (TGEV),  Mycoplasma hyopneumoniae , Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), Infectious Bursal Disease Virus (IBDV), Parvovirus, Poxvirus, Rotavirus, and Influenza Virus; and further wherein when the translocation domain is the Stx translocation peptide, the antigen is located at the N-terminal of the fusion protein; and when the translocation domain is the PE translocation peptide, the CD40-binding domain is located at the N-terminal of the fusion protein.   
     
     
         19 . The fusion protein of  claim 18 , wherein the CD40L or the functional fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 17, 18 and 19. 
     
     
         20 . A method for eliciting an antigen-specific, cell-mediated immune response, or for reducing, inhibiting, treating, and/or ameliorating an infectious animal disease caused by a pathogen in an animal in need thereof, comprising:
 administering a therapeutically effective amount of the fusion protein of  claim 18  to the animal in need thereof, and thereby eliciting the antigen-specific, cell-mediated immune response, or reducing, inhibiting, treating, and/or ameliorating the infectious animal disease caused by the pathogen in the animal in need thereof.   
     
     
         21 . The fusion protein of  claim 1 , wherein the antigen is a fusion antigen comprising at least two antigenic polypeptides. 
     
     
         22 . The fusion protein of  claim 1 , wherein the antigen is selected from the group consisting of ASFV CP204L protein, ASFV E183L protein, a fusion antigen of ASFV CP204L and E183L, and a fusion antigen of CSFV E2 and NS3p.